The rearrangement of nitrogen-fixation (nif) genes during heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120 offers a unique example of an environmentally induced cellular differentiation involving programmed genome rearrangement. When filaments of Anabaena are grown in the absence of a combined nitrogen source, approximately 10% of the vegetative cells evenly spaced along the filament form heterocysts, terminally differentiated cells responsible for nitrogen fixation. Heterocyst development involves many morphological and biochemical changes including activation of the nif genes and repression of the genes for oxygen-evolving photosystem II and carbon fixation. Two different genome rearrangements occur near the nif genes of Anabaena during heterocyst differentiation: the excision of an 11-kb element from within the nifD gene, and the excision of a 55-kb element from within the fdxN gene. Both elements interrupt nif operons in vegetative cells and excise from the chromosome late during heterocyst differentiation. The long-term objective of this project is to understand gene regulation during heterocyst differentiation. More specifically, we are interested in the developmental regulation of the DNA rearrangements, the rearrangement mechanisms, and the role played by the rearrangements in heterocyst development and function. We are also interested in using the DNA rearrangements as tools to identify new genes involved in the regulation of gene expression during heterocyst differentiation. The specific aims of the proposed research are as follows: 1. Analysis of the developmental regulation of the xisA gene which is involved in the nifD 11-kb element excision. Both genetic and biochemical approaches will be employed to study the recently identified upstream regulatory region and the xisA promotor. Genes affecting xisA expression will be identified. 2. The identification and characterization of the gene(s) required for the fdxN 55-kb rearrangement including the gene for the site-specific recombinase. A genetic selection for mutations affecting rearrangement of the 55-kb element will be developed which will use a rearrangement substrate plasmid. 3. Analysis of the developmental regulation of the nifD 11-kb and fdxN 55-kb rearrangements with the goal of understanding the developmental coordination between the two rearrangements and the linkage between the DNA rearrangements and induction of the nif genes. 4. Determination of the properties and functions of the 11-kb and 55-kb elements in vegetative cells and heterocysts of Anabaena. Genetic analysis of the DNA elements will use conjugal expression vectors and our recently described methods for chromosomal gene inactivation in Anabaena.